Targeting 4-1BB and PD-L1 induces potent and durable antitumor immunity in B-cell lymphoma

IntroductionAlthough PD-1/L1 mAb has demonstrated clinical benefits in certain cancer types, low response rate and resistance remain the main challenges for the application of these immune checkpoint inhibitors (ICIs). 4-1BB is a co-stimulator molecule expressed in T cells, which could enhance T cell proliferation and activation. Herein, the synergetic antitumor effect and underlying mechanism of 4-1BB agonist combined with PD-1/PD-L1 blockade were determined in B-cell lymphoma (BCL).MethodsSubcutaneous transplantation BCL tumor models and metastasis models were established to evaluate the therapeutic effect of PD-L1 antibody and/or 4-1BB agonist in vivo. For the mechanistic study, RNA-seq was applied to analyze the tumor microenvironment and immune-related signal pathway after combination treatment. The level of IFN-γ, perforin, and granzyme B were determined by ELISA and Real-time PCR assays, while tumor-infiltrating T cells were measured by flow cytometry and immunohistochemical analysis. CD4/CD8 specific antibodies were employed to deplete the related T cells to investigate the role CD4+ and CD8+ T cells played in combination treatment.ResultsOur results showed that combining anti-PD-L1 ICI and 4-1BB agonists elicited regression of BCL and significantly extended the survival of mice compared to either monotherapy. Co-targeting PD-L1 and 4-1BB preferentially promoted intratumoral cytotoxic lymphocyte infiltration and remodeled their function. RNA-sequence analysis uncovered a series of up-regulated genes related to the activation and proliferation of cytotoxic T lymphocytes, further characterized by increased cytokines including IFN-γ, granzyme B, and perforin. Furthermore, depleting CD8+ T cells not CD4+ T cells totally abrogated the antitumor efficacy, indicating the crucial function of the CD8+ T cell subset in the combination therapy.DiscussionIn summary, our findings demonstrated that 4-1BB agonistic antibody intensified the antitumor immunity of anti-PD-1/PD-L1 ICI via promoting CD8+ T cell infiltration and activation, providing a novel therapeutic strategy to BCL

Hexokinase 2 (HK2), the tumor promoter in glioma, is downregulated by miR-218/Bmi1 pathway

In cancer, glycolysis driving enzymes and their regulating microRNAs are one of the key focus of oncology research lately. The glycolytic enzyme hexokinase 2 (HK2) is crucial for the Warburg effect in human glioma, the most common malignant brain tumor. In the present study, we studied the tumorigenic role of HK2 in glioma, and clarified the mechanism of miR-218 induced HK2 regulation in glioma development. The HK2 expression in patient derived glioma and non neoplastic brain tissue was quantified. The HK2 silenced U87 and U251 cell lines were assessed for their proliferation, migration and invasive potential in vitro, while the tumor forming potential of U87 cells was evaluated in vivo. The untreated cell lines served as control. The HK2 expression in (a) lentivirus-infected, miR-218 overexpressing and (b) shRNA mediated Bmi1 silenced U87 and U251 glioma cell lines were quantified. Luciferase reporter assay, qRT-PCR analysis and WB were employed as required. The HK2 expression was significantly increased in glioma tissues comparing with the non neoplastic brain tissues and was positively correlated with the glioma grade. Silencing HK2 in glioma cell lines significantly decreased their proliferation, migration, invasion and tumorigenic abilities. Although, overexpression of miR-218 significantly downregulated the HK2 expression, luciferase reporter assay failed to show HK2 as the direct target of miR-218. A direct correlation, however, was observed between HK2 and Bmi-1, the direct target of miR-218. Taken together, our findings confirmed the tumorigenic activity of HK2 in glioma, and the involvement of the miR218/Bmi1 pathway in the regulation of its expression

SOX9-PDK1 axis is essential for glioma stem cell self-renewal and temozolomide resistance

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor with limited therapeutic options. Glioma stem cell (GSC) is thought to be greatly responsible for glioma tumor progression and drug resistance. But the molecular mechanisms of GSC deriving recurrence and drug resistance are still unclear. SOX9 (sex-determining region Y (SRY)-box9 protein), a transcription factor expressed in most solid tumors, is reported as a key regulator involved in maintaining cancer hallmarks including the GSCs state. Previously, we have observed that silencing of SOX9 suppressed glioma cells proliferation both in vitro and in vivo. Here, we found that SOX9 was essential for GSC self-renewal. Silencing of SOX9 down-regulated a broad range of stem cell markers and inhibited glioma cell colony and sphere formation. We identified pyruvate dehydrogenase kinase 1 (PDK1) as a target gene of SOX9 using microarray analyses. PDK1 inactivation greatly inhibited glioma cell colony and sphere formation and sensitized glioma spheres to temozolomide (TMZ) toxicity. In addition, SOX9-shRNA and PDK1 inhibitor could greatly sensitize GSC to TMZ in vivo. Taken together, our data reveals that SOX9-PDK1 axis is a key regulator of GSC self-renewal and GSC temozolomide resistance. These findings may provide help for future human GBM therapy

MicroRNA-101 inhibits proliferation, migration and invasion of human glioblastoma by targeting SOX9

Glioblastoma multiforme (GBM) is the most common primary malignant tumors originating in the brain parenchyma. At present, GBM patients have a poor prognosis despite the continuous progress in therapeutic technologies including surgery, radiotherapy, photodynamic therapy, and chemotherapy. Recent studies revealed that miR-101 was remarkably down-regulated in kinds of human cancers and was associated with aggressive tumor cell proliferation and stem cell self-renewal. Data also showed that miR-101 was down-regulated in primary glioma samples and cell lines, but the underlying molecular mechanism of the deregulation of miR-101 in glioma remained largely unknown. In this study, we found that miR-101 could inhibit the proliferation and invasion of glioma cells both in vitro and in vivo by directly targeting SOX9 [sex-determining region Y (SRY)-box9 protein]. Silencing of SOX9 exerted similar effects with miR-101 overexpression on glioma cells proliferation and invasion. Quantitative reverse transcription PCR and Western blotting analysis revealed a negative relationship between miR-101 and SOX9 in human glioma U251MG and U87MG cells, and the luciferase assay indicated that miR-101 altered SOX9 expression by directly targeting on 3′UTR. Taken together, our findings suggest that miR-101 regulates glioma proliferation, migration and invasion via directly down-regulating SOX9 both in vitro and in vivo, and miR-101 may be a potential therapeutic target for future glioma treatment

A novel fusion protein consisting of anti-ANGPTL3 antibody and interleukin-22 ameliorates diabetic nephropathy in mice

IntroductionThe pathogenic mechanisms of diabetic nephropathy (DN) include podocyte injury, inflammatory responses and metabolic disorders. Although the antagonism of Angiopoietin-like protein 3 (ANGPTL3) can alleviate proteinuria symptoms by inhibiting the activation of integrin αvβ3 on the surface of podocytes, it can not impede other pathological processes, such as inflammatory responses and metabolic dysfunction of glucolipid. Interleukin-22 (IL-22) is considered to be a pivotal molecule involved in suppressing inflammatory responses, initiating regenerative repair, and regulating glucolipid metabolism.MethodsGenes encoding the mIL22IgG2aFc and two chains of anti-ANGPTL3 antibody and bifunctional protein were synthesized. Then, the DN mice were treated with intraperitoneal injection of normal saline, anti-ANGPTL3 (20 mg/kg), mIL22Fc (12 mg/kg) or anti-ANGPTL3 /IL22 (25.3 mg/kg) and irrigation of positive drug losartan (20mg/kg/d) twice a week for 8 weeks.ResultsIn this research, a novel bifunctional fusion protein (anti-ANGPTL3/IL22) formed by the fusion of IL-22 with the C-terminus of anti-ANGPTL3 antibody exhibited favorable stability and maintained the biological activity of anti-ANGPTL3 and IL-22, respectively. The fusion protein showed a more pronounced attenuation of proteinuria and improved dysfunction of glucolipid metabolism compared with mIL22Fc or anti-ANGPTL3. Our results also indicated that anti-ANGPTL3/IL22 intervention significantly alleviated renal fibrosis via inhibiting the expression of the inflammatory response-related protein nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) p65 and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome. Moreover, transcriptome analysis revealed the downregulation of signaling pathways associated with injury and dysfunction of the renal parenchymal cell indicating the possible protective mechanisms of anti-ANGPTL3/IL22 in DN.ConclusionCollectively, anti-ANGPTL3/IL22 bifunctional fusion protein can be a promising novel therapeutic strategy for DN by reducing podocyte injury, ameliorating inflammatory response, and enhancing renal tissue recovery

Systematic Review of MicroRNAs and its Therapeutic Potential in Glioma

MicroRNAs (miRNAs) are short noncoding RNAs. The discovery of miRNA has provided a novel tool to the research of tumor pathogenesis, and a new strategy to the diagnosis and prognosis of human cancers. Currently, numerous studies have indicated that the deregulation of miRNAs in glioma is closely related to glioma pathogenesis and progress. miRNAs function as key regulators of glioma through negative control of the target gene expression, by targeting the 3′-untranslated region of its messenger RNA which regulate the cell proliferation, apoptosis and prognosis of glioma. Moreover, radiation and chemotherapy resistance in glioma therapy is also caused by deregulation of miRNAs. It has been suggested that miRNAs act as tumor suppressors or oncogenes in glioma. Not only can miRNAs be used as biomarkers of glioma diagnosis and therapy, but also as novel targets of glioma gene therapy

The mechanism of BMI1 in regulating cancer stemness maintenance, metastasis, chemo- and radiation resistance

BMI1 is involved in the occurrence and development of many types of cancer through a variety of signaling pathways. BMI1, which is overexpressed in cancer, is often associated with chemo- and radiation resistance and poor prognosis in cancer patients. This article reviews the current understanding of the mechanism of BMI1 in maintaining tumor stemness, promoting metastasis, and inducing chemo- and radiation resistance, aiming at providing updated information supportive of targeting BMI1 in cancer treatment

ClC-3 Expression and Its Association with Hyperglycemia Induced HT22 Hippocampal Neuronal Cell Apoptosis

Although apoptosis plays an important role in the development of Diabetic Encephalopathy (DE), the underlying molecular mechanisms remain unclear. With respect to this, the present work aims to study the variation in chloride/proton exchanger ClC-3 expression and its association with HT22 hippocampal neuronal apoptosis under hyperglycemic condition in vitro. The cells were stimulated with added 0, 5, or 25 mM glucose or mannitol for up to 72 hours before assessing the rate of ClC-3 expression, cell viability, and apoptosis. In a consecutive experiment, cells received chloride channel blocker in addition to glucose. The rate of cellular death/apoptosis and viability was measured using Flow Cytometry and MTT assay, respectively. Changes in ClC-3 expression were assessed using immunofluorescence staining and western blot analysis. The results revealed a significant increase in cellular apoptosis and reduction in viability, associated with increased ClC-3 expression in high glucose group. Osmolarity had no role to play. Addition of chloride channel blocker completely abolished this effect. Thus we conclude that, with its increased expression, ClC-3 plays a major role in hyperglycemia induced hippocampal neuronal apoptosis. To strengthen our understanding of this aforesaid association, we conducted an extensive literature search which is presented in this paper

A novel indication of thioredoxin-interacting protein as a tumor suppressor gene in malignant glioma

Malignant glioma, the most common form of primary brain tumor, is associated with substantial morbidity and mortality, owing to the lack of response shown by patients to conventional therapies. Additional therapeutic targets and effective treatment options for these patients are therefore required. In the present study, a possible association of thioredoxin-interacting protein (TXNIP) with malignant glioma was evaluated. Initially, semi-quantitative and quantitative analysis of the expression levels of TXNIP in clinical specimens of primary glioma was performed via immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, and expression levels were further correlated to the overall survival time of the patients. The proliferative, migratory and invasive properties of the glioblastoma U251 cell line, engineered to downregulate TXNIP by lentiviral transfection of a specific short hairpin RNA, were evaluated by means of in vitro assays. Consequently, IHC and RT-qPCR analysis revealed a negative association between the expression level of TXNIP and the histopathological grade of the tumor. Higher TXNIP expression level was associated with extended patient survival time. In vitro analysis revealed increased growth, migration and invasion in U251 cells with downregulated TXNIP expression compared with their non-transfected counterparts. These findings strongly indicate that TXNIP functions as a tumor suppressor in malignant glioma cells and underscore its potential as a novel therapeutic target and prognostic indicator of the condition

Activating Autophagy Enhanced the Antitumor Effect of Antibody Drug Conjugates Rituximab-Monomethyl Auristatin E

BackgroundAntibody drug conjugate (ADC) showed potent therapeutic efficacy in several types of cancers. The role of autophagy in antitumor effects of ADC remains unclear.MethodsIn this study, the ADC, Rituximab-monomethyl auristatin E (MMAE) with a Valine–Citrulline cleavable linker, was designed to investigate its therapeutic efficacy against non-Hodgkin lymphoma (NHL) as well as the underlying mechanisms. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect growth inhibition in B-cell lymphoma cell lines, Ramos and Daudi cells, which were treated by Rituximab-MMAE alone or combined with autophagy conditioner. Apoptosis was detected by flow cytometry and immunohistochemistry, and apoptosis inhibitor was employed to discover the relationship between autophagy and apoptosis during the Rituximab-MMAE treatment. Autophagy was determined by three standard techniques which included confocal microscope, transmission electron microscope, and western blots. Ramos xenograft tumors in BALB/c nude mice were established to investigate the antitumor effect of combination use of Rituximab-MMAE and autophagy conditioner in B-NHL therapy.ResultsOur results showed that Rituximab-MMAE elicited caspase-3-dependent apoptosis in NHL cells and exhibited potent therapeutic efficacy in vivo. Autophagy, which was characterized by upregulated light chain 3-II expression, and accumulation of autophagosomes, was triggered during the Rituximab-MMAE treatment. Meanwhile, inactivation of Akt/mTOR pathway was shown to be involved in the autophagy triggered by Rituximab-MMAE, indicating a probable mechanism of the ADC-initiated autophagy. Importantly, inhibition of autophagy by chloroquine suppressed the Rituximab-MMAE-induced apoptosis, while activating autophagy by rapamycin significantly enhanced the therapeutic effect of Rituximab-MMAE both in vitro and in vivo.ConclusionOur data elucidated the critical relationship between autophagy and apoptosis in Rituximab-MMAE-based therapy and highlighted the potential approach for NHL therapy by combined administration of the ADC and autophagy activator